Herpes simplex virus proteins synthesized in infected cells change in both number and character as the infection proceeds. Underlying these changes in viral proteins are changes in the species and abundance of the viral mRNAs. We proposed that the time of appearance of early and late HSV mRNA is regulated predominantly by the ability to initiate transcription and that several factors control this process. These include primarily DNA sequences upstream from the promotor and viral regulatory proteins. Our goal is to identify the regulatory proteins and the DNA sequences with which they interact. To assay for upstream control elements in viral DNA we will construct recombinant plasmids containing isolated HSV genes, modify them by mutational insertions or deletions and assay their activity in vitro and in vivo. To study transcriptional regulation from these genes, we will use a three-pronged approach that measures RNA synthesis in nuclear extracts, in isolated nuclei, and in cells following transfection with plasmid DNA. The results from the in vivo experiments will be used to assess the validity of the in vitro measurements. Taken together, the data will establish whether transcription of HSV genes is regulated by viral proteins acting either independently or in a coordinate manner and whether specific gene sequences are involved. Long term goals are to study the biochemical interactions between the viral proteins and the enzymes involved in RNA synthesis. The availability of an in vitro transcription system makes the biochemical approach possible.